Anti-chlamydial methods and materials

ABSTRACT

The present invention relates to methods for treating chlamydial infection comprising administering to a subject suffering from a chlamydial infection a bactericidal permeability-inducing (BPI) protein product.

This is a Continuation of U.S. application Ser. No. 09/281,985, filedMar. 29, 1999, now U.S. Pat. No. 6,162,788, which is a Continuation ofU.S. application Ser. No. 08/694,843, filed Aug. 9, 1996, now U.S. Pat.No. 5,888,973.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods of treatingchlamydial infections by administration ofbactericidal/permeability-increasing (BPI) protein products.

BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. HumanBPI protein has been isolated from PMNs by acid extraction combined witheither ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000(1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652(1987)]. BPI obtained in such a manner is referred to herein as naturalBPI and has been shown to have potent bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein and the nucleic acid sequence of DNAencoding the protein have been reported in FIG. 1 of Gray et al., J.Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al. amino acid sequence is set out in SEQ ID NO: 1 hereto.

BPI is a strongly cationic protein. The N-terminal half of BPI accountsfor the high net positive charge; the C-terminal half of the moleculehas a net charge of −3. [Elsbach and Weiss (1981), supra.] A proteolyticN-terminal fragment of BPI having a molecular weight of about 25 kD hasan amphipathic character, containing alternating hydrophobic andhydrophilic regions. This N-terminal fragment of human BPI possesses theanti-bacterial efficacy of the naturally-derived 55 kD human BPIholoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. Incontrast to the N-terminal portion, the C-terminal region of theisolated human BPI protein displays only slightly detectableanti-bacterial activity against gram-negative organisms. [Ooi et al., J.Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately23 kD, referred to as “rBPI₂₃,” has been produced by recombinant meansand also retains anti-bacterial activity against gram-negativeorganisms. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).

The bactericidal effect of BPI has been reported to be highly specificto gram-negative species, e.g., in Elsbach and Weiss, Inflammation:Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter30, Raven Press, Ltd. (1992). This reported target cell specificity wasbelieved to be the result of the strong attraction of BPI forlipopolysaccharide (LPS), which is unique to the outer membrane (orenvelope) of gram-negative organisms. Although BPI was commonly thoughtto be non-toxic for other microorganisms, including yeast, and forhigher eukaryotic cells, it has recently been discovered that BPIprotein products, as defined infra, exhibit activity againstgram-positive bacteria, mycoplasma, mycobacteria, fungi, and protozoa.[See allowed, co-owned, co-pending U.S. patent application Ser. No.08/372,783 filed Jan. 13, 1995, the disclosures of which areincorporated herein by reference; co-owned, copending U.S. patentapplication Ser. No. 08/626,646, the disclosures of which areincorporated herein by reference; co-owned, co-pending U.S. patentapplication Ser. No. 08/372,105, the disclosures of which areincorporated herein by reference; and co-owned, co-pending U.S. patentapplication Ser. No. 08/273,470, the disclosures of which areincorporated herein by reference.] It has also been discovered that BPIprotein products have the ability to enhance the activity of antibioticsagainst bacteria. [See U.S. Pat. No. 5,523,288, the disclosures of whichare incorporated herein by reference, and allowed, co-owned, co-pendingU.S. patent application Ser. No. 08/372,783.]

The precise mechanism by which BPI kills gram-negative bacteria is notyet completely elucidated, but it is believed that BPI must first bindto the surface of the bacteria through electrostatic and hydrophobicinteractions between the cationic BPI protein and negatively chargedsites on LPS. LPS has been referred to as “endotoxin” because of thepotent inflammatory response that it stimulates, i.e., the release ofmediators by host inflammatory cells which may ultimately result inirreversible endotoxic shock. BPI binds to lipid A, reported to be themost toxic and most biologically active component of LPS.

In susceptible gram-negative bacteria, BPI binding is thought to disruptLPS structure, leading to activation of bacterial enzymes that degradephospholipids and peptidoglycans, altering the permeability of thecell's outer membrane, and initiating events that ultimately lead tocell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act intwo stages. The first is a sublethal stage that is characterized byimmediate growth arrest, permeabilization of the outer membrane andselective activation of bacterial enzymes that hydrolyze phospholipidsand peptidoglycans. Bacteria at this stage can be rescued by growth inserum albumin supplemented media [Mannion et al., J. Clin. Invest.,85:853-860 (1990)]. The second stage, defined by growth inhibition thatcannot be reversed by serum albumin, occurs after prolonged exposure ofthe bacteria to BPI and is characterized by extensive physiologic andstructural changes, including apparent damage to the inner cytoplasmicmembrane.

Initial binding of BPI to LPS leads to organizational changes thatprobably result from binding to the anionic groups of LPS, whichnormally stabilize the outer membrane through binding of Mg⁺⁺ and Ca⁺⁺.Attachment of BPI to the outer membrane of gram-negative bacteriaproduces rapid permeabilization of the outer membrane to hydrophobicagents such as actinomycin D. Binding of BPI and subsequentgram-negative bacterial killing depends, at least in part, upon the LPSpolysaccharide chain length, with long O-chain bearing, “smooth”organisms being more resistant to BPI bactericidal effects than shortO-chain bearing, “rough” organisms [Weiss et al., J. Clin. Invest. 65:619-628 (1980)]. This first stage of BPI action, permeabilization of thegram-negative outer envelope, is reversible upon dissociation of theBPI, a process requiring high concentrations of divalent cations andsynthesis of new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)].Loss of gram-negative bacterial viability, however, is not reversed byprocesses which restore the envelope integrity, suggesting that thebactericidal action is mediated by additional lesions induced in thetarget organism and which may be situated at the cytoplasmic membrane(Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specificinvestigation of this possibility has shown that on a molar basis BPI isat least as inhibitory of cytoplasmic membrane vesicle function aspolymyxin B (In't Veld et al., Infection and Immunity 56: 1203-1208(1988)) but the exact mechanism as well as the relevance of suchvesicles to studies of intact organisms has not yet been elucidated.

Chlamydia are nonmotile, gram-negative, obligate intracellular bacteriathat have unusual biological properties which phylogeneticallydistinguish them from other families of bacteria. Chlamydiae arepresently placed in their own order, the Chlamydiales, familyChlamydiaceae, with one genus, Chlamydia. [Schachter and Stamm,Chlamydia, in Manual of Clinical Microbiology, pages 669-677, AmericanSociety for Microbiology, Washington, D.C. (1995).] There are fourspecies, Chlamydia trachomatis, C. pneumoniae, C. psittaci and C.pecorum, which cause a wide spectrum of human diseases. In developingcountries, C. trachomatis causes trachoma, the world's leading cause ofpreventable blindness. Over 150 million children have active trachoma,and over 6 million people are currently blind from this disease. Inindustrialized countries, C. trachomatis is the most prevalent sexuallytransmitted disease, causing urethritis, cervicitis, epididymitis,ectopic pregnancy and pelvic inflammatory disease. Last year alone, anestimated 300 million people contracted sexually transmitted chlamydialinfections. Among the 250,000 cases of pelvic inflammatory disease peryear in the United States, approximately 25,000 women are renderedinfertile each year. Neonatal C. trachomatis infections, contracted atbirth from infected mothers, cause hundreds of thousands ofconjunctivitis cases per year, of which about half of these infectedinfants develop pneumonia. Recently, C. pneumoniae has been implicatedas a common cause of epidemic human pneumonitis. Members of the genusare not only important human pathogens, but also cause significantmorbidity in other mammals and birds. Thus, chlamydia are one of themost ubiquitous pathogens in the animal kingdom. [Zhang et al., Cell,69:861-869 (1992).]

Their unique developmental cycle differentiates them from all othermicroorganisms. They are obligate intracellular parasites that areunable to synthesize ATP, and thus depend on the host cells' energy tosurvive. Unlike viruses, they always contain both DNA and RNA, divide bybinary fission, contain ribosomes, and can synthesize proteins.Chlamydia have cell walls similar in structure to those of gram-negativebacteria, and all members of the genus carry a unique LPS-like antigen,termed complement fixation (CF) antigen, that may be analogous to theLPS of certain gram-negative bacteria. [Schachter and Stamm, supra.]Chlamydia also carry a major outer membrane protein (MOMP) that containsboth species and subspecies-specific antigens.

The infectious form of chlamydia is the elementary body (EB), whichinfects mammalian cells by attaching to the host cell and entering in ahost-derived phagocytic vesicle (endosome), within which the entiregrowth cycle is completed. The target host cell in vivo is typically thecolumnar epithelial cell, and the primary mode of entry is believed tobe receptor-mediated endocytosis. Once the EB has entered the cell, itreorganizes into a reticulate body (RB) that is larger than the EB andmetabolically active, synthesizing DNA, RNA and proteins. The EBs arespecifically adapted for extracellular survival, while the metabolicallyactive RBs do not survive well outside the host cell and seems adaptedfor an intracellular milieu. After approximately 8 hours, the RBs begindividing by binary fission. As they replicate within the endosomes ofhost cells, they form characteristic intracellular inclusions that canbe seen by light microscopy. After a period of growth and division, theRBs reorganize and condense to form infectious EBs. The developmentalcycle is complete when host cell lysis or exocytosis of chlamydiaoccurs, releasing the EBs to initiate another cycle of infection. Thelength of the complete developmental cycle, as studied in cell culturemodels, is 48 to 72 hours and varies as a function of the infectingstrain, host cell and environmental conditions. [Beatty et al.,Microbiol. Rev., 58(4):686-699 (1994).]

It has been demonstrated, at least for C. trachomatis, that attachmentof the chlamydia organism to host cells is mediated by a heparansulfate-like glycosaminoglycan (GAG) present on the surface of thechlamydia. Treatment of chlamydia with either purified heparin, heparinsulfate, or heparin receptor analogs (such as platelet factor 4 andfibronectin, both of which are known to bind heparin sulfate), inhibitedthe attachment and infectivity of chlamydia to host cells. Inhibitionwas not seen with non-heparin GAGs, such as hyaluronate, chondroitinsulfate, or keratin sulfate. Treatment of C. trachomatis withheparitinase reduced attachment and infectivity by greater than 90%;subsequent treatment with exogenous heparan sulfate was able to restorethe ability of treated organisms to attach to host cells in adose-dependent manner. Other GAGs such as hyaluronate, chondroitinsulfate, or keratin sulfate did not restore attachment ability. Thesedata suggest that a heparin sulfate-like GAG mediates attachment ofchlamydia to host cells by bridging mutual GAG receptors on the hostcell surface and on the chlamydial outer membrane surface. [Zhang etal., Cell, 69:861-869 (1992).]

C. trachomatis is almost exclusively a human pathogen, and isresponsible for trachoma, inclusion conjunctivitis, lymphogranulomavenereum (LGV), and genital tract diseases. [Schachter and Stamm,supra.] Within this species, serotypes A, B, Ba, and C have beenassociated with endemic trachoma, the most common preventable form ofblindness in the world. Trachoma is a chronic inflammation of theconjunctiva and the cornea, which is not sexually transmitted. Thepotentially blinding sequelae of trachoma include lid distortion,trichiasis (misdirection of lashes), and entropion (inward deformationof the lid margin). These can cause corneal ulceration followed by lossof vision. Serotypes L1, L2, and L3 of C. trachomatis are associatedwith LGV. Untreated, lymphogranuloma venereum progresses through threestages, each more severe than the preceding one. The primary lesion, ifpresent, appears on the genitals. The second stage is a bubonic statemarked by regional lymphadenopathy, during which the buboes maysuppurate and develop draining fistulas. Rectal strictures and lymphaticobstruction can appear in the tertiary stage. Lymphogranuloma venereumis a common problem in developing countries with tropical or subtropicalclimates, especially among the lower socioeconomic groups.

C. trachomatis is also the most common agent of sexually transmitteddisease. In men, serotypes D through K are the major identifiable causesof nongonococcal urethritis, and also cause epididymitis, Reiter'ssyndrome, and proctitis. Chlamydial infections are not easily identifiedin men by clinical symptoms alone, because the infection may beasymptomatic and because other pathogens cause similar symptoms.Chlamydial urethritis occurs twice as frequently as gonococcalurethritis (gonorrhea) in some populations, and its incidence is on theincrease. Even when N. gonorrhea is shown to be present, the urethritismay be due to a dual or multiple infection involving a second organism.Concurrent C. trachomatis and N. gonorrhoea infections have beenreported in about 25 percent of men with gonorrhea. Epididymitis is themost important complication of chlamydial urethritis in men. C.trachomatis causes one of every two cases of epididymitis in younger menin the United States, with sterility a possible result. Reiter'ssyndrome is another manifestation of chlamydial infection in men. It isa painful systemic illness that classically includes symptoms ofurethritis, conjunctivitis and arthritis. Urethritis and arthritis areby far the most frequent combination; it appears that the chlamydialurethral infection may trigger the arthritis. C. trachomatis can alsocause proctitis (anal inflammation), particularly in homosexual men.

In women, chlamydial infection with the sexually transmitted serotypesresults in cervicitis, urethritis, endometritis, salpingitis, andproctitis; serious sequelae of salpingitis include tubal scarring,infertility, and ectopic pregnancy. Unrecognized chlamydial infectionsin women are common. Approximately 50 percent of women infected withchlamydia are asymptomatic. C. trachomatis causes mucopurulentcervicitis and the urethral syndrome, as well as endometritis andsalpingitis. These upper genital tract chlamydial infections may causesterility or predispose to ectopic pregnancies and are the gravestcomplications of chlamydial infections in women. Ten percent of allmaternal deaths are due to ectopic pregnancies. C. trachomatis causesover 30 percent of the cases of mucopurulent cervicitis. As many asone-half of the women with gonococcal cervicitis have a concomitantchlamydial infection. If the gonococcal infection is treated withpenicillin, the concomitant chlamydial cervicitis will continueundetected and untreated, and may progress to pelvic inflammatorydisease (salpingitis), which can lead to sterility and ectopicpregnancies. C. trachomatis is a cause of the urethral syndrome inwomen. Chlamydial infections may ascend from the cervix to theendometrium, where C. trachomatis has been found in the epitheliallining of the uterine cavity. It is estimated that about one-half of allwomen will cervicitis have endometritis. Salpingitis, a major cause ofectopic pregnancies and infertility, is the most serious complication offemale genital infections. Upper abdominal pain is the predominantsymptom of perihepatitis. Both C. trachomatis and N. gonorrhoea cancause perihepatitis. This condition occurs almost exclusively in womenin whom the infecting organisms spread to the surface of the liver frominflamed fallopian tubes.

Women infected with C. trachomatis may also pass the disease to theirnewborn as it passes through the infected birth canal. These newbornsmost often develop inclusion conjunctivitis or chlamydial pneumonia, butmay also develop vaginal, pharyngeal, or enteric infections. Though notblinding, inclusion conjunctivitis can become chronic, causing mildscarring and pannus formulation if left untreated. During passagethrough the birth canal, up to two-thirds of babies born to mothers withchlamydial genital infections will also become infected. With as many asone in ten pregnant women having chlamydial cervicitis in some parts ofthe world, the risk to newborns is considerable. Chlamydial pneumoniaoccurs in 10 percent to 20 percent of infants born to infected mothers.C. trachomatis is responsible for 20 percent to 60 percent of allpneumonias during the first 6 months of life.

C. trachomatis strains are sensitive to the action of tetracyclines,macrolides and sulfonamides and produce a glycogen-like material withinthe inclusion vacuole that stains with iodine.

C. psittaci strains infect many avian species and mammals, producingsuch diseases as psittacosis, ornithosis, feline pneumonitis, and bovineabortion. [Schachter and Stamm, supra.] C. psittaci is ubiquitous amongavian species, and infection in birds usually involves the intestinaltract. The organism is shed in the feces, contaminates the environment,and is spread by aerosol. C. psittaci is also common in domesticmammals. In some parts of the world, these infections have importanteconomic consequences, as C. psittaci is a cause of a number of systemicand debilitating diseases in domestic mammals and, most important, cancause abortions. Human chlamydial infections from this agent usuallyresult from exposure to an infected avian species, but may also occurafter exposure to infected domestic mammals. This species is resistantto the action of sulfonamides and produces inclusions that do not stainwith iodine.

C. pneumoniae has less than 10% DNA relatedness to the other species andhas pear-shaped rather than round elementary bodies (EBs). Like C.trachomatis, it appears to be exclusively a human pathogen without ananimal reservoir. C. pneumoniae has been identified as the cause of avariety of respiratory tract diseases and is distributed worldwide.[Schachter and Stamm, supra.] Infections appear to be commonly acquiredin later childhood, adolescence, and early adulthood, resulting inseroprevalences of 40 to 50% in 30 to 40-year-old people. Manifestationsof infection include pharyngitis, bronchitis, and mild pneumonia, andtransmission is primarily via respiratory secretions. Inseroepidemiological studies, these infections have been linked withcoronary artery disease, and their role in atherosclerosis is currentlyunder intense scrutiny.

The role of C. pecorum as a pathogen is not clear, and specializedreagents are required for its identification.

The recommended procedure for primary isolation of chlamydia is cellculture. Chlamydia will grow in the yolk sac of the embryonated hen egg,as well as in cell culture (with some variability). C. trachomatis caninfect several cell lines, such as McCoy's heteroploid murine cells,HeLa 229 cells, BHK-21 cells, or L-929 cells. HL cells and Hep-2 cellsmay be more sensitive for the recovery of C. pneumoniae. The most commontechnique involves inoculation of clinical specimens intocycloheximide-treated McCoy cells. The basic principle involvescentrifugation of the inoculum onto the cell monolayer, incubation ofthe monolayers for 48 to 72 hours, and demonstration of typicalintracytoplasmic inclusions by appropriate immunofluorescence, iodine orGiemsa staining procedures. Cell culture generally requires two to sixdays to complete because of the incubation time required.

Chlamydia may also be detected in samples by the direct fluorescentantibody (DFA) test, in which slides are incubated withfluorescein-conjugated monoclonal antibodies, and fluorescing elementarybodies are detected using a fluorescent microscope. This test hasapproximately 80% to 90% sensitivity and 98% to 99% specificity comparedwith cell cultures when both tests are performed under idealcircumstances. [Schachter and Stamm, supra.]

A number of commercially available products can detect chlamydialantigens in clinical specimens by using enzyme immunoassay (EIA)procedures. Most of these products detect chlamydial LPS, which is moresoluble than MOMP. Without confirmation, the tests have a specificity onthe order of 97%. [Schachter and Stamm, supra.] Several nucleic acidprobes are also commercially available. One commercially available probetest (GenProbe) utilizes DNA-RNA hybridization in an effort to increasesensitivity by detecting chlamydial RNA.

The complement fixation (CF) test is the most frequently performedserological test, and measures serum level of complement-fixing antibody(antibody to the group CF antigen). It is useful for diagnosingpsittacosis, in which paired acute- and convalescent-phase sera oftenshow four-fold or greater increases in titer. The same seems to be truefor many C. pneumoniae infections. Approximately 50% of these infectionsare CF-positive, although it may take 24 weeks to detect seroconversion.CF testing may also be useful in diagnosing LGV, in which single-pointtiters greater than 1:64 are highly supportive of this clinicaldiagnosis. [Schachter and Stamm, supra.] High titers ofcomplement-fixing antibodies are not found in chlamydial conjunctivitisor genital tract infections, and therefore are not sensitive for theseinfections.

The microimmunofluorescence (micro-IF) method is a much more sensitiveprocedure for measuring anti-chlamydial antibodies. This indirectfluorescent antibody technique uses antigens prepared by infecting theyolk sacs of fertile chick embryos with each chlamydial serotype. Serialdilutions of patient serum are added to the prepared antigens, and thelevel of antibody in the blood sample is determined with the use ofimmunofluorescence. Trachoma, inclusion conjunctivitis, and genitaltract infections may be diagnosed by the micro-IF technique ifappropriately timed paired sera can be obtained, but the procedure is oflimited clinical utility because diagnosis requires demonstration of afour-fold or greater change in antibody titer in paired specimens, andbecause patients with superficial genital infections such as urethritismay not have a change in titer. However, a high antibody titer in asingle serum specimen from a patient with Reiter's syndrome and a highIgM titer in the serum of an infant with pneumonia are helpful inestablishing a diagnosis.

Strain-to-strain variation in antimicrobial susceptibility profiles andnewly acquired drug resistance are both very infrequent among chlamydia.Among the drugs most active in vitro against C. trachomatis, C.pneumoniae, and C. psittaci are the tetracyclines, such as tetracyclineand doxycycline, the macrolides, such as erythromycin and azithromycin,the quinolones, such as ciprofloxacin and ofloxacin, chloramphenicol,rifampin, clindamycin and the sulfonamides. The tetracyclines andmacrolides have generally been the mainstays of therapy for infectionsdue to chlamydia. [Schachter and Stamm, supra; Goodman and Gilman, ThePharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, New York,N.Y. (1996).]

Antimicrobial susceptibility testing is infrequently performed forchlamydial infections, but may be conducted as follows. The organismsfor testing are grown for at least two passages in cells cultured inantibiotic-free media before being harvested. An adjusted inoculum of˜100 inclusion-forming units per microtiter well is then used to infectantibiotic-free cell monolayers. After centrifugation of the inoculumonto the monolayer, serial dilutions of the test antibiotic can be addedeither immediately or at various time intervals over the next 24 hours.After 48 hours, fluorescein-conjugated monoclonal antibodies are use toidentify minimum inhibitory concentration (MIC), i.e., the highestantibiotic dilution that inhibits intracellular inclusion formation.Generally, monolayers are also disrupted and further passaged to definethe minimum bactericidal concentration (MBC), i.e., the highestantibiotic dilution that prevents viable chlamydia from being detectedin passage (MBC).

SUMMARY OF THE INVENTION

The present invention provides methods of treating a subject sufferingfrom a chlamydial infection by administering a therapeutically effectiveamount of a BPI protein product. This is based on the surprisingdiscoveries that BPI protein products inhibit the infectivity ofchlamydia and inhibit the proliferation of chlamydia in an establishedintracellular infection. The BPI protein products may be administeredalone or in conjunction with other known anti-chlamydial agents. Whenmade the subject of adjunctive therapy, the administration of BPIprotein products may reduce the amount of non-BPI anti-chlamydial agentneeded for effective therapy, thus limiting potential toxic responseand/or high cost of treatment. Administration of BPI protein productsmay also enhance the effect of such agents, accelerate the effect ofsuch agents, or reverse resistance of chlamydia to such agents.

In addition, the invention provides a method of killing or inhibitinggrowth of chlamydia comprising contacting the chlamydia with a BPIprotein product. This method can be practiced in vivo or in a variety ofin vitro uses such as use to decontaminate fluids and surfaces and tosterilize surgical and other medical equipment and implantable devices,including prosthetic joints and indwelling invasive devices.

A further aspect of the invention involves use of a BPI protein productfor the manufacture of a medicament for treatment of chlamydialinfection. The medicament may include, in addition to a BPI proteinproduct, other chemotherapeutic agents such as non-BPI anti-chlamydialagents.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon considering the followingdetailed description of the invention, which describes the presentlypreferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the surprising discovery that a BPIprotein product can be administered to treat subjects suffering fromchlamydial infection, and provides methods of prophylactically ortherapeutically treating such infections. Unexpectedly, BPI proteinproducts were demonstrated to have anti-chlamydial activities, asmeasured, for example, by a reduction in the number of reproductivebodies seen in the host cells. A variety of chlamydial infections,including infections caused by C. trachomatis, C. pneumoniae, C.psittaci and C. pecorum, may be treated according to the invention.

The term “treating” or “treatment” as used herein encompasses bothprophylactic and therapeutic treatment.

The BPI protein product may be administered systemically or topically.Systemic routes of administration include oral, intravenous,intramuscular or subcutaneous injection (including into depots forlong-term release), intraocular or retrobulbar, intrathecal,intraperitoneal (e.g. by intraperitoneal lavage), transpulmonary usingaerosolized or nebulized drug, or transdermal. Topical routes includeadministration in the form of salves, creams, jellies, ophthalmic dropsor opthalmic ointments, ear drops, suppositories, such as vaginalsuppositories, or irrigation fluids (for, e.g., irrigation of wounds).

When given parenterally, BPI protein product compositions are generallyinjected in doses ranging from 1 μg/kg to 100 mg/kg per day, preferablyat doses ranging from 0.1 mg/kg to 20 mg/kg per day, and more preferablyat doses ranging from 1 to 20 mg/kg/day. The treatment may continue bycontinuous infusion or intermittent injection or infusion, or acombination thereof, at the same, reduced or increased dose per day foras long as determined by the treating physician. When given topically,BPI protein product compositions are generally applied in unit dosesranging from 1 μg/mL to 1 gm/mL, and preferably in doses ranging from 1μg/mL to 100 mg/mL. Those skilled in the art can readily optimizeeffective dosages and monotherapeutic or concurrent administrationregimens for BPI protein product and/or other anti-chlamydial agents, asdetermined by good medical practice and the clinical condition of theindividual patient.

The BPI protein product may be administered in conjunction with otheranti-chlamydial agents presently known to be effective. Preferredanti-chlamydial agents for this purpose include the tetracyclines, suchas tetracycline and doxycycline, the macrolides, such as erythromycinand azithromycin, the quinolones, such as ciprofloxacin and ofloxacin,chloramphenicol, rifampin, clindamycin and the sulfonamides. Concurrentadministration of BPI protein product with anti-chlamydial agents isexpected to improve the therapeutic effectiveness of the anti-chlamydialagents. This may occur through reducing the concentration ofanti-chlamydial agent required to eradicate or inhibit chlamydialgrowth, e.g., replication. Because the use of some agents is limited bytheir systemic toxicity or prohibitive cost, lowering the concentrationof anti-chlamydial agent required for therapeutic effectiveness reducestoxicity and/or cost of treatment, and thus allows wider use of theagent. Concurrent administration of BPI protein product and anotheranti-chlamydial agent may produce a more rapid or complete bactericidalor bacteriostatic effect than could be achieved with either agent alone.BPI protein product administration may reverse the resistance ofchlamydia to anti-chlamydial agents. BPI protein product administrationmay also convert a bacteriostatic agent into a bactericidal agent.

An advantage of the present invention is that the wide spectrum ofactivity of BPI protein products against a variety of organisms, and theuse of BPI protein products as adjunctive therapy to enhance theactivity of antibiotics makes BPI protein products an excellent choicefor treating dual or multiple infections with chlamydia and anotherorganism, such as the gram-negative bacteria N. gonorrhea. Thus, BPIprotein products may be especially useful in inhibiting transmission ofsexually transmitted diseases, which often involve dualgonococcal/chlamydial infection. It is therefore contemplated that BPIprotein products will be incorporated into contraceptive compositionsand devices, e.g., included in spermicidal creams or jellies, or coatedon the surface of condoms.

Another advantage is the ability to treat chlamydia that have acquiredresistance to known anti-chlamydial agents. A further advantage ofconcurrent administration of BPI with an anti-chlamydial agent havingundesirable side effects is the ability to reduce the amount ofanti-chlamydial agent needed for effective therapy. The presentinvention may also provide quality of life benefits due to, e.g.,decreased duration of therapy, reduced stay in intensive care units orreduced stay overall in the hospital, with the concomitant reduced riskof serious nosocomial (hospital-acquired) infections.

“Concurrent administration” as used herein includes administration ofthe agents together, or before or after each other. The BPI proteinproducts and anti-chlamydial agents may be administered by differentroutes. For example, the BPI protein product may be administeredintravenously while the anti-chlamydial agents are administeredintramuscularly, intravenously, subcutaneously, orally orintraperitoneally. Alternatively, the BPI protein product may beadministered intraperitoneally while the anti-chlamydial agents areadministered intraperitoneally or intravenously, or the BPI proteinproduct may be administered in an aerosolized or nebulized form whilethe anti-chlamydial agents are administered, e.g., intravenously. TheBPI protein product and anti-chlamydial agents may be both administeredintravenously. The BPI protein product and anti-chlamydial agents may begiven sequentially in the same intravenous line, after an intermediateflush, or may be given in different intravenous lines. The BPI proteinproduct and anti-chlamydial agents may be administered simultaneously orsequentially, as long as they are given in a manner sufficient to allowboth agents to achieve effective concentrations at the site ofinfection.

Concurrent administration of BPI protein product and antibiotic isexpected to provide more effective treatment of chlamydial infections.Concurrent administration of the two agents may provide greatertherapeutic effects in vivo than either agent provides when administeredsingly. For example, concurrent administration may permit a reduction inthe dosage of one or both agents with achievement of a similartherapeutic effect. Alternatively, the concurrent administration mayproduce a more rapid or complete bactericidal/bacteriostatic effect thancould be achieved with either agent alone.

Therapeutic effectiveness is based on a successful clinical outcome, anddoes not require that the anti-chlamydial agent or agents kill 100% ofthe organisms involved in the infection. Success depends on achieving alevel of anti-chlamydial activity at the site of infection that issufficient to inhibit the chlamydia in a manner that tips the balance infavor of the host. When host defenses are maximally effective, theanti-chlamydial effect required may be minimal. Reducing organism loadby even one log (a factor of 10) may permit the host's own defenses tocontrol the infection. In addition, augmenting an earlybactericidal/bacteriostatic effect can be more important than long-termbactericidal/bacteriostatic effect. These early events are a significantand critical part of therapeutic success, because they allow time forhost defense mechanisms to activate.

BPI protein product is thought to interact with a variety of hostdefense elements present in whole blood or serum, including complement,p15 and LBP, and other cells and components of the immune system. Suchinteractions may result in potentiation of the activities of BPI proteinproduct. Because of these interactions, BPI protein products can beexpected to exert even greater activity in vivo than in vitro. Thus,while in vitro tests are predictive of in vivo utility, absence ofactivity in vitro does not necessarily indicate absence of activity invivo. For example, BPI has been observed to display a greaterbactericidal effect on gram-negative bacteria in whole blood or plasmaassays than in assays using conventional media. [Weiss et al., J. Clin.Invest. 90:1122-1130 (1992)]. This may be because conventional in vitrosystems lack the blood elements that facilitate or potentiate BPI'sfunction in vivo, or because conventional media contain higher thanphysiological concentrations of magnesium and calcium, which aretypically inhibitors of the activity of BPI protein products.Furthermore, in the host, BPI protein product is available to neutralizetranslocation of gram-negative bacteria and concomitant release ofendotoxin, a further clinical benefit not seen in or predicted by invitro tests.

It is also contemplated that the BPI protein product be administeredwith other products that potentiate the activity of BPI proteinproducts, including the anti-chlamydial activity of BPI proteinproducts. For example, serum complement potentiates the gram-negativebactericidal activity of BPI protein products; the combination of BPIprotein product and serum complement provides synergisticbactericidal/growth inhibitory effects. See, e.g., Ooi et al. J. Biol.Chem., 265: 15956 (1990) and Levy et al. J. Biol. Chem., 268: 6038-6083(1993) which address naturally-occurring 15 kD proteins potentiating BPIantibacterial activity. See also co-owned, co-pending PCT ApplicationNo. US94/07834 filed Jul. 13, 1994, which corresponds to U.S. patentapplication Ser. No. 08/274,303 filed Jul. 11, 1994 as acontinuation-in-part of U.S. patent application Ser. No. 08/093,201filed Jul. 14, 1993. These applications, which are all incorporatedherein by reference, describe methods for potentiating gram-negativebactericidal activity of BPI protein products by administeringlipopolysaccharide binding protein (LBP) and LBP protein products. LBPprotein derivatives and derivative hybrids which lack CD-14immunostimulatory properties are described in PCT Application No.US94/06931 filed Jun. 17, 1994, which corresponds to co-owned,co-pending U.S. patent application Ser. No. 08/261,660, filed Jun. 17,1994 as a continuation-in-part of U.S. patent application Ser. No.08/079,510, filed Jun. 17, 1993, the disclosures of all of which arehereby incorporated by reference. It has also been observed thatpoloxamer surfactants enhance the anti-bacterial activity of BPI proteinproducts as described in Lambert, U.S. application Ser. No. 08/586,133filed Jan. 12, 1996, which is a continuation-in-part of U.S. applicationSer. No. 08/530,599 filed Sep. 19, 1995, which is a continuation-in-partof U.S. application Ser. No. 08/372,104 filed Jan. 13, 1995, all ofwhich correspond to PCT Application No. PCT/US96/01095; poloxamersurfactants may also enhance the activity of anti-chlamydial agents.

In addition, the invention provides a method of killing or inhibitinggrowth of chlamydia comprising contacting the chlamydia with a BPIprotein product. This method can be practiced in vivo or in a variety ofin vitro uses such as to decontaminate fluids and surfaces or tosterilize surgical and other medical equipment and implantable devices,including prostheses and intrauterine devices. These methods can also beused for in situ sterilization of indwelling invasive devices such asintravenous lines and catheters, which are often foci of infection.

A further aspect of the invention involves use of a BPI protein productfor the manufacture of a medicament for treatment of chlamydialinfection. The medicament may include, in addition to a BPI proteinproduct, other chemotherapeutic agents such as anti-chlamydial agents.The medicament can optionally comprise a pharmaceutically acceptablediluent. adjuvant or carrier.

As used herein, “BPI protein product” includes naturally andrecombinantly produced BPI protein, natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs; and BPI-derived peptides. TheBPI protein products administered according to this invention may begenerated and/or isolated by any means known in the art. U.S. Pat. No.5,198,541, the disclosure of which is incorporated herein by reference,discloses recombinant genes encoding, and methods fc expression of, BPIproteins including recombinant BPI holoprotein, referred to as rBPI andrecombinant fragments of BPI. Co-owned, copending U.S. patentapplication Ser. No. 07/885,501 and a continuation-in-part thereof, U.S.patent application Ser. No. 08/072,063 filed May 19, 1993 andcorresponding PCT Application No. 93/04752 filed May 19, 1993, which areall incorporated herein by reference, disclose novel methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transformed mammalian host cells in cultureand discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same or similar amino acidsequence as a natural human BPI holoprotein, except that the fragmentmolecule lacks amino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein. Nonlimiting examples ofsuch fragments include a N-terminal fragment of natural human BPI ofapproximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649(1991), and the recombinant expression product of DNA encodingN-terminal amino acids from 1 to about 193 to 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI)has also been produced having the sequence (SEQ ID NOS: 145 and 146) setout in FIG. 1 of Gray et al., supra, with the exceptions noted forrBPI₂₃ and with the exception that residue 417 is alanine (specified byGCT) rather than valine (specified by GTT). Other examples includedimeric forms of BPI fragments, as described in co-owned and co-pendingU.S. patent application Ser. No. 08/212,132, filed Mar. 11, 1994, andcorresponding PCT Application No. PCT/US95/03125, the disclosures ofwhich are incorporated herein by reference. Preferred dimeric productsinclude dimeric BPI protein products wherein the monomers areamino-terminal BPI fragments having the N-terminal residues from about 1to 175 to about 1 to 199 of BPI holoprotein. A particularly preferreddimeric product is the dimeric form of the BPI fragment havingN-terminal residues 1 through 193, designated rBPI₄₂ dimer.

Biologically active variants of BPI (BPI variants) include but are notlimited to recombinant hybrid fusion proteins, comprising BPIholoprotein or biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed by Theofan et al. in co-owned, copending U.S. patentapplication Ser. No. 07/885,911, and a continuation-in-part applicationthereof, U.S. patent application Ser. No. 08/064,693 filed May 19, 1993and corresponding PCT Application No. US93/04754 filed May 19, 1993,which are all incorporated herein by reference and include hybrid fusionproteins comprising, at the amino-terminal end, a BPI protein or abiologically active fragment thereof and, at the carboxy-terminal end,at least one constant domain of an immunoglobulin heavy chain or allelicvariant thereof.

Biologically active analogs of BPI (BPI analogs) include but are notlimited to BPI protein products wherein one or more amino acid residueshave been replaced by a different amino acid. For example, co-owned,copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, thedisclosures of which are incorporated herein by reference, disclosespolypeptide analogs of BPI and BPI fragments wherein a cysteine residueis replaced by a different amino acid. A stable BPI protein productdescribed by this application is the expression product of DNA encodingfrom amino acid 1 to approximately 193 or 199 of the N-terminal aminoacids of BPI holoprotein, but wherein the cysteine at residue number 132is substituted with alanine and is designated rBPI₂₁ Δcys or rBPI₂₁.Other examples include dimeric forms of BPI analogs; e.g. co-owned andco-pending U.S. patent application Ser. No. 08/212,132 filed Mar. 11,1994, and corresponding PCT Application No. PCT/US95/03125, thedisclosures of which are incorporated herein by reference.

Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced byrecombinant or synthetic means (BPI-derived peptides), such as thosedescribed in co-owned and copending PCT Application No. US94/10427 filedSep. 15, 1994, which corresponds to U.S. patent application Ser. No.08/306,473, filed Sep. 15, 1994, and PCT Application No. US94/02465filed Mar. 11, 1994, which corresponds to U.S. patent application Ser.No. 08/209,762, filed Mar. 11, 1994, which is a continuation-in-part ofU.S. patent application Ser. No. 08/183,222, filed Jan. 14, 1994, whichis a continuation-in-part of U.S. patent application Ser. No. 08/093,202filed Jul. 15, 1993 (for which the corresponding internationalapplication is PCT Application No. US94/02401 filed Mar. 11, 1994),which is a continuation-in-part of U.S. patent application Ser. No.08/030,644 filed Mar. 12, 1993, the disclosures of all of which areincorporated herein by reference.

Presently preferred BPI protein products include recombinantly-producedN-terminal fragments of BPI, especially those having a molecular weightof approximately between 21 to 25 kD such as rBPI₂₁ or rBPI₂₃, ordimeric forms of these N-terminal fragments (e.g., rBPI₄₂ dimer).Additionally, preferred BPI protein products include rBPI andBPI-derived peptides.

The administration of BPI protein products is preferably accomplishedwith a pharmaceutical composition comprising a BPI protein product and apharmaceutically acceptable diluent, adjuvant, or carrier. The BPIprotein maybe administered without or in conjunction with knownsurfactants, other chemotherapeutic agents or additional knownantichlamydial agents. A stable pharmaceutical composition containingBPI protein products (e.g., rBPI, rBPI₂₃) comprises the BPI proteinproduct at a concentration of 1 mg/ml in citrate buffered saline (5 or20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight ofpoloxamer 188 (PLURONIC F-68. BASF Wyandotte, Parsippany, N.J.) and0.002% by weight of polysorbate 80 (TWEEN 80, ICI Americas Inc.,Wilmington, Del.). Another stable pharmaceutical composition containingBPI protein products (e.g., rBPI₂₁) comprises the BPI protein product ata concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer188 and 0.002% polysorbate 80. Such preferred combinations are describedin co-owned, co-pending PCT Application No. US94/01239 filed Feb. 2,1994, which corresponds to U.S. patent application Ser. No. 08/190,869filed Feb. 2, 1994 and U.S. patent application Ser. No. 08/012,360 filedFeb. 2, 1993, the disclosures of all of which are incorporated herein byreference.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples. Example 1addresses the use of BPI protein product to inhibit infection of hostcells with chlamydia when administered at the same time as chlamydialchallenge. Example 2 addresses the anti-chlamydial activity of BPIprotein product in chlamydia-infected host cells.

EXAMPLE 1 Use of BPI Protein Product to Inhibit Infection of Host Cellswith Chlamydia

A. Preparation of Chlamydia Stock

Chlamydia trachomatis (Ct) serovar L2 stock was prepared as follows.McCoy cells (ATCC Accession No. CRL 1696) were cultured overnight ingrowth medium [Eagles Medium Nutrient Mixture (MEM), M-3786, Sigma, St.Louis, Mo.] with 1 sodium pyruvate (S-8636, Sigma) and 10% fetal bovineserum (FBS. A115-L, Hyclone, Logan, Vt.). The media was aspirated and avial of Ct was rapidly thawed and mixed with 30 mL of Dulbecco'sphosphate buffered saline (PBS, Sigma) and 7% sucrose (DPBS-7). Ten mLof the suspension were added to each of 3 T150 flasks and the flaskswere incubated at 37° C. while being rocked periodically over the nexttwo hours to distribute the inoculum. The DPBS-7 was aspirated from theflasks and 50 mL of growth media were added to each flask. Afterincubation for three days at 37° C. in 5% CO₂, the Ct was harvested asfollows. The growth media was aspirated from the flasks and glass beadswere added to the flasks to a depth of ˜0.25 inches. Ten mL Eagles MEM(without FBS) was added to each flask and the beads were rocked over themonolayer until all the cells were dislodged. The beads and cell debriswere collected in 50 mL screw-capped centrifuge tubes, the flasks werewashed twice with PBS, and the washings were added to the beadsuspension. Each tube was placed on ice and sonicated for 60 seconds todisrupt the cells. The disrupted cells/bead suspension were centrifugedat low speed (˜800 rpm). The supernatant was removed and collected in a250 mL polycarbonate centrifuge bottle, then centrifuged for one hour athigh speed (˜25,000×g). The pellet was resuspended in FBS (40 mL) byrepeated passage through a #16 gauge needle and syringe. One mL aliquotswere distributed into NUNC® (Naperville, Ill.) cryovials and frozen at−70° C.

B. Titration of Chlamydia Stock

Three vials of Ct stock prepared as described above in Section A wererapidly thawed at 37° C. and serially diluted in 10-fold concentrationsin Eagles MEM or DPBS-7 without serum. Twenty-four well plates withcoverslips in each well containing 24-hour McCoy cell monolayers wereprepared. The media was aspirated, the wells were washed once with PBS,and 1 mL of each Ct dilution in either Eagles MEM or DPBS-7 was added toquadruplicate sets of McCoy cells. The plates were incubated at 37° C.in 5% CO₂ for 2 hours, the media was aspirated and 2 mL of growth mediawas added. The plates were then reincubated at 27° C. in 5% CO₂ for 3days, fixed in methanol, and stained for 30 minutes in a moist chamberwith an FITC-labelled mouse monoclonal anti-chlamydia antibody (SyvaMicroTrak® Chlamydia trachomatis Culture Confirmation Test). The stainedcoverslips were washed in water, air dried, inverted into a drop ofmounting fluid (50% glycerol; 50% PBS) and viewed using a Leitzfluorescent microscope with a 25X objective (excitation wavelength 480nm, emission wavelength 520 nm). The inclusion bodies were counted andcomparable results were obtained over the 10⁻² to 10⁻¹⁰ concentrationrange tested in the Eagles MEM and DPBS-7. The 10⁻⁵ dilution of thestock preparation gave 100-300 inclusion body-forming units/mL; thisdilution was selected for use in all subsequent studies using this Ctstock. Additional media studies were performed using Basal Medium Eagle(BME, Sigma), Eagles MEM (E-MEM, Sigma), RPMI-1640 with HEPES (Sigma),RPMI-1640 without HEPES (Sigma), F-12 (Gibco) and Dulbecco's ModifiedEagle's Medium Nutrient Mixture F-12 Ham (DMEM/F-12, Gibco). DMEM/F-12without FBS was selected for use in subsequent Chlamydia infectivitystudies. Media without FBS was selected for use because the addition of10% FBS to the above tested media inhibited infection of McCoy cells byCt.

C. Infection by Chlamydia in the Presence or Absence of BPI ProteinProduct

The BPI protein product tested was rBPI₂₁ [2 mg/mL in 5 mM sodiumcitrate, 150 mM sodium chloride, pH 5.0, with 0.2% PLURONIC® P123 (BASFWyandotte, Parsippany, N.J.), 0.002% polysorbate 80 (TWEEN® 80, ICIAmericas Inc., Wilmington, Del.) and 0.05% EDTA]. Equal volumes offormulation buffer alone [5 mM sodium citrate, 150 mM sodium chloride,pH 5.0, with 0.2% PLURONIC® P123, 0.002% polysorbate 80 and 0.05% EDTA]were used as a control. Serial dilutions of rBPI₂₁ or formulation bufferwere prepared with DMEM/F-12 (without FBS) so that when the serialdilutions were added at a 9:1 ratio to 1 mL of a 10⁻⁴ dilution of Ctstock, the final concentration of Ct would be a 10⁻⁵ dilution of Ctstock and the final rBPI₂₁ concentrations would be 128, 64, 32, 16 and 8μg/mL. Comparable (by volume) formulation buffer controls were alsoprepared. The final suspensions were incubated at 37° C. for 30 minutesin a water bath.

McCoy cells in DMEM/F-12/10%FBS were seeded at 2×10⁵ cells/well into24-well tissue culture plates (Corning #25820), incubated for 24 hoursand the media aspirated. Cr, with and without BPI, was added in 1 mL toduplicate wells at each rBPI₂₁ concentration. The plates werecentrifuged at 2500 rpm for 30 minutes, incubated for 2 hours at 37° C.in 5% CO₂, and the wells aspirated. Each well received 2 mL ofDMEM/F-12/10%FBS and 1 μg/mL cycloheximide (Sigma) and the platesreincubated for 3 days. After removal of the media, the wells werewashed with phosphate buffered saline (PBS), air dried, fixed withmethanol and stained with Gram's iodine. The cells may be alternativelystained with FITC-labelled anti-chlamydia antibodies as described insection B above.

Using an inverted microscope, 100% of each well was scanned for thepresence of inclusion bodies, which stain brown with Gram's iodine dueto the high concentration of glycogen in vacuoles produced by thereproductive bodies. Results are shown below in Table 1.

TABLE 1 Number of Inclusion Bodies per Well with rBPI₂₁ without rBPI₂₁rBPI₂₁ Concentration (mean of 4 wells) (value for 1 well) 128 μg/mL 0110  64 μg/mL 0 115  32 μg/mL 0 115  16 μg/mL 1.5 114  8 μg/mL 59 124Positive Control 151 (Ct only) Negative Control  0 (no Ct)

These representative results from one of three studies indicate thatrBPI₂ can inhibit infection of permissive cells.

EXAMPLE 2 Anti-Chlamydial Activity of BPI Protein Product AgainstChlamydia-Infected Host Cells

Chlamydia trachomatis (Ct) serovar L2 stock prepared as described inExample 1 was diluted to 10⁻⁵ with Dulbecco's Modified Eagle's MediumNutrient Mixture F-12 Ham (DMEM/F-12) with 10% fetal bovine serum (FBS).

McCoy cells in DMEM/F-12/10%FBS were seeded at 1×10⁵ cells/well into24-well tissue culture plates (Corning #25820), incubated for 24 hours,and the media aspirated. Ct (1 mL of the 10⁻⁵ stock) was added to eachwell of four plates except for two negative control wells per plate. Theplates were centrifuged at 2500 rpm for 30 minutes, incubated for 24hours at 37° C. in 5% CO₂, and the wells aspirated.

rBPI₂₁ as described in Example 1 was diluted to final concentrations of128, 64, 32, 16 and 8 μg/mL in DMEM/F-12 and 1.0 mL added to theappropriate duplicate wells on each plate. Comparable formulation buffercontrols as described in Example 1 were also prepared. The plates wereincubated for 2 hours, and 1 mL of DMEM/F-12/20%FBS and 2 μg/mLcycloheximide was added to all wells, causing the rBPI₂₁ concentrationto decrease by a factor of two. The plates were reincubated for up to 5days.

At 24, 48, 72 and 120 hours, the media was removed from a single plate,the wells washed with PBS and air dried, fixed with methanol and stainedwith Gram's iodine. Using an inverted microscope, 100% of each well wasscanned for the presence of inclusion bodies. Results are shown in Table2 below.

TABLE 2 Initial RBPI₂₁ Number of Inclusion Bodies Per WellConcentration* at 24 hours at 48 hours at 72 hours  0 285.5 398 335.75 8 194.5 180 108 16 138 140.5 109.5 32 112.5 95 57.5 64 119.5 81 39 128 113 77.5 5 *This initial concentration, which was present for the firsttwo hours of incubation, was decreased to half of the initial value forthe remainder of the 5-day incubation.

These representative results from one of two studies show that rBPI₂₁ atinitial concentrations ranging from 16 μg/mL to 128 μg/mL was able toreduce the number of intracellular inclusion bodies in Ct-infected cellswhen administered 24 hours after challenge with Ct.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description on the presently preferred embodimentsthereof. Consequently the only limitations which should be placed uponthe scope of the present invention are those that appear in the appendedclaims.

2 1813 base pairs nucleic acid single linear cDNA CDS 31..1491mat_peptide 124..1491 misc_feature “rBPI” 1 CAGGCCTTGA GGTTTTGGCAGCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC 54 Met Arg Glu Asn Met AlaArg Gly -31 -30 -25 CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTCGTC GCC ATA 102 Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu ValAla Ile -20 -15 -10 GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTGGTC AGG ATC 150 Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val ValArg Ile -5 1 5 TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCCGCT CTG 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala AlaLeu 10 15 20 25 CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TCA GACAGC TTT 246 Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp SerPhe 30 35 40 AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATGGAC 294 Lys Ile Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp45 50 55 ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT342 Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Ile Ser Met Val Pro Asn 6065 70 GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATC AAG ATC AGC GGG390 Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile Lys Ile Ser Gly 7580 85 AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGC AAT TTT GAC438 Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 9095 100 105 CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTG AAG CTG GGCAGT 486 Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser110 115 120 AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TGC TCC AGC TGC AGCAGC 534 Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser125 130 135 CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC AAA GTC GGGTGG 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser Lys Val Gly Trp140 145 150 CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCG CTT CGA AACAAG 630 Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys155 160 165 ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCCAAG 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys170 175 180 185 CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA ATG ACC AAA ATAGAT TCT 726 Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met Thr Lys Ile AspSer 190 195 200 GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CCT CCA GCA ACCACG GCT 774 Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr ThrAla 205 210 215 GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TTT TAC AGT GAGAAC CAC 822 Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu AsnHis 220 225 230 CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG ATG GAG TTT CCCGCT GCC 870 His Asn Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro AlaAla 235 240 245 CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GAC TAC TTC TTCAAC ACA 918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe AsnThr 250 255 260 265 GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TTG AAG ATGACC CTT AGA 966 Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met ThrLeu Arg 270 275 280 GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CGA CTG ACAACC AAG TTC 1014 Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Arg Leu Thr ThrLys Phe 285 290 295 TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AAG TTT CCCAAC ATG AAG 1062 Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Lys Phe Pro AsnMet Lys 300 305 310 ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CCA CAC CTGTCT GTG CAG 1110 Ile Gln Ile His Val Ser Ala Ser Thr Pro Pro His Leu SerVal Gln 315 320 325 CCC ACC GGC CTT ACC TTC TAC CCT GCC GTG GAT GTC CAGGCC TTT GCC 1158 Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln AlaPhe Ala 330 335 340 345 GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TTC CTGATT GGC ATG CAC 1206 Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu IleGly Met His 350 355 360 ACA ACT GGT TCC ATG GAG GTC AGC GCC GAG TCC AACAGG CTT GTT GGA 1254 Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn ArgLeu Val Gly 365 370 375 GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CTG AAGCAC TCA AAT ATT 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys HisSer Asn Ile 380 385 390 GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT ATC ATGAAC TAC ATT GTA 1350 Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Ile Met AsnTyr Ile Val 395 400 405 CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AAA CTACAG AAA GGC TTC 1398 Pro Ile Leu Val Leu Pro Arg Val Asn Glu Lys Leu GlnLys Gly Phe 410 415 420 425 CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TACAAC GTA GTG CTT CAG 1446 Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr AsnVal Val Leu Gln 430 435 440 CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GACGTT GTC TAT AAA 1491 Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val ValTyr Lys 445 450 455 TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTGTTCCTGATGG GCTGTGGGGC 1551 ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAACCAAGAGCCC CTTGCAAACT 1611 TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAACATTATTCAT TGGAAAAGTG 1671 CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGGCTAAGGCTGC AGAGATATTT 1731 CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCCATTTGTGCTT CATGAAAAAA 1791 AACTTCTGGT TTTTTTCATG TG 1813 487 amino acidsamino acid linear protein 2 Met Arg Glu Asn Met Ala Arg Gly Pro Cys AsnAla Pro Arg Trp Val -31 -30 -25 -20 Ser Leu Met Val Leu Val Ala Ile GlyThr Ala Val Thr Ala Ala Val -15 -10 -5 1 Asn Pro Gly Val Val Val Arg IleSer Gln Lys Gly Leu Asp Tyr Ala 5 10 15 Ser Gln Gln Gly Thr Ala Ala LeuGln Lys Glu Leu Lys Arg Ile Lys 20 25 30 Ile Pro Asp Tyr Ser Asp Ser PheLys Ile Lys His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met AspIle Arg Glu Phe Gln Leu Pro Ser 50 55 60 65 Ser Gln Ile Ser Met Val ProAsn Val Gly Leu Lys Phe Ser Ile Ser 70 75 80 Asn Ala Asn Ile Lys Ile SerGly Lys Trp Lys Ala Gln Lys Arg Phe 85 90 95 Leu Lys Met Ser Gly Asn PheAsp Leu Ser Ile Glu Gly Met Ser Ile 100 105 110 Ser Ala Asp Leu Lys LeuGly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120 125 Ile Thr Cys Ser SerCys Ser Ser His Ile Asn Ser Val His Val His 130 135 140 145 Ile Ser LysSer Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 150 155 160 Ile GluSer Ala Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys 165 170 175 ValThr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 180 185 190Pro Val Met Thr Lys Ile Asp Ser Val Ala Gly Ile Asn Tyr Gly Leu 195 200205 Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys 210215 220 225 Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe AlaPro 230 235 240 Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val TyrLeu Gly 245 250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val TyrGln Glu Ala 260 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met IlePro Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly ThrPhe Leu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe Pro Asn Met Lys IleGln Ile His Val Ser Ala Ser 310 315 320 Thr Pro Pro His Leu Ser Val GlnPro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val Asp Val Gln Ala PheAla Val Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser Leu Phe Leu Ile GlyMet His Thr Thr Gly Ser Met Glu Val Ser 355 360 365 Ala Glu Ser Asn ArgLeu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370 375 380 385 Leu Glu LeuLys His Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu 390 395 400 Gln AspIle Met Asn Tyr Ile Val Pro Ile Leu Val Leu Pro Arg Val 405 410 415 AsnGlu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val 420 425 430Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe 435 440445 Gly Ala Asp Val Val Tyr Lys 450 455

What is claimed are:
 1. A method of treating chlamydial infectionscomprising concurrent administration to a subject suffering from achlamydial infection of a bactericidal/permeability-increasing (BPI)protein product and a non-BPI anti-chlamydial agent effective to treatthe chlamydial infection.
 2. The method of claim 1 wherein the BPIprotein product is BPI holoprotein, rBPI₂₃ or rBPI₂₁.
 3. The method ofclaim 1 wherein the BPI protein product is administered at a dose ofbetween 1 μg/kg to 100 mg/kg per day.
 4. The method of claim 1 whereinthe BPI protein product is administered topically at a dose of between 1μg/mL to 1 gm/mL.
 5. The method of claim 1 wherein the BPI proteinproduct comprises an N-terminal fragment of BPI having a molecularweight of about 21 kD to 25 kD.
 6. The method of claim 5 wherein the BPIprotein product is a dimer comprising the N-terminal fragment having amolecular weight of about 21 kD to 25 kD.
 7. The method of claim 1wherein the chlamydial infection comprises a chlamydial species selectedfrom the group consisting of C. trachomans, C. pneumoniae, C. psittaci,and C. pecorum species.
 8. The method of claim 7 wherein the chlamydialspecies is C. trachomatis.
 9. The method of claim 1 wherein the non-BPIanti-chlamydial agent is a tetracycline, a macrolide, a quinolone,chloramphenicol, rifampin, clindamycin or a sulfonamide.
 10. A method ofkilling or inhibiting replication of chlamydia comprising concurrentcontacting of the chlamydia with a bactericidal/permeability-increasing(BPI) protein product and a non-BPI anti-chlamydial agent, wherein thenon-BPI anti-chlamydial agent is a tetracycline, a macrolide, aquinolone, chloramphenicol, rifampin, clindamycin or a sulfonamide. 11.The method of claim 10 wherein the tetracycline is tetracycline ordoxycycline.
 12. The method of claim 9 or 10 wherein the macrolide iserythromycin or aztihromycin.
 13. The method of claim 9 or 10 whereinthe quinolone is ciprofloxacin or ofloxacin.
 14. The method of claim 9or 10 wherein chlamydia intracellular inclusion bodies in infected hostcells are reduced in number.
 15. The method of claim 1 or 9 wherein thesubject is suffering from coronary artery disease.
 16. The method ofclaim 10 wherein surgical equipment, implantable devices or indwellinginvasive devices are covered with the BPI protein product and thenon-BPI anti-chlamydial agent.
 17. The method of claim 16 wherein theimplantable devices are prostheses or intrauterine evices.
 18. Themethod of claim 16 wherein the indwelling invasive devices areintravenous lines or catheters.